ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

ABCA1 and scavenger receptor class B,type I,are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells

The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB.     

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.     

Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands

ATP-binding cassette transporter-1 (ABCA1) is a cause of Tangier disease, which is a familial deficiency of plasma high density lipoproteins (HDL). This molecule is known to be expressed in the multiple tissues and organs including small intestines, liver, and macrophages in the blood vessels. Recent in vivo studies suggested that ABCA1 plays some roles in the flux of cholesterol in the intestines. One of the major questions to understand the roles of ABCA1 in the intestines is the expression pattern in the intestinal epithelial cells. To address this issue, we have investigated the expression and regulation of ABCA1 in Caco-2 cells cultured on Transwell as a model, especially focusing on possible polarized expression of ABCA1. The expression of ABCA1 was up-regulated during the differentiation and under the stimulation of LXR/RXR by the addition of 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-OH). Apolipoprotein-AI-mediated cholesterol efflux was dominant toward the basolateral side of polarized cells when stimulated by 9-cis-RA and 22-OH. The cell surface biotinylation experiment followed by Western blot analyses demonstrated a markedly dominant expression of ABCA1 on the basolateral surface, which was clearly confirmed by the confocal laser scanning microscopy. In conclusion, the present study demonstrates that ABCA1 is dominantly expressed on the basolateral surface of Caco-2 cells tested, suggesting that this molecule may play a role in the basolateral movement of cholesterol at least when stimulated by LXR/RXR ligands.     

Model system for the analysis of cell surface expression of human ABCA1

Background  

The ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of HDL particles and removing cellular cholesterol. Both the proper expression of ABCA1 in the plasma membrane and the internalization along with apoA-I are required for function. Therefore, we developed a model system to investigate the effect of clinically relevant drugs on the cell surface appearance of ABCA1.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

The role of the ABCA1 transporter and cholesterol efflux in familial hypoalphalipoproteinemia

Defects in the gene encoding for the ATP binding cassette (ABC) transporter A1 (ABCA1) were shown to be one of the genetic causes for familial hypoalphalipoproteinemia (FHA). We investigated the role of ABCA1-mediated cholesterol efflux in Dutch subjects suffering from FHA. Eighty-eight subjects (mean HDL cholesterol levels 0.63 +/- 0.21 mmol/l) were enrolled. Fibroblasts were cultured and loaded with [3H]cholesterol. ABCA1 and non-ABCA1-mediated efflux was studied by using apolipoprotein A-I (apoA-I), HDL, and methyl-beta-cyclodextrin as acceptors. Efflux to apoA-I was decreased in four patients (4/88, 4.5%), and in all cases, a mutation in the ABCA1 gene was found. In the remaining 84 subjects, no correlation between efflux and apoA-I or HDL cholesterol was found. Efflux to both HDL and cyclodextrin, in contrast, did correlate with HDL cholesterol plasma levels (r = 0.34, P = 0.01; and r = 0.27, P = 0.008, respectively). The prevalence of defects in ABCA1-dependent cholesterol efflux in Dutch FHA patients is low. The significant correlation between plasma HDL cholesterol levels and methyl-beta-cyclodextrin-mediated efflux in the FHA patients with normal ABCA1 function suggests that non-ABCA1-mediated efflux might also be important for plasma HDL cholesterol levels in these individuals.     

Regulatory effects of synthetic liver X receptor- and peroxisome-proliferator activated receptor agonists on sterol transport pathways in polarized cerebrovascular endothelial cells

The blood-brain barrier contributes to maintain brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. Brain capillary endothelial cells, representing a physiological barrier to the central nervous system, express apolipoprotein A-I (apoA-I, the major high-density lipoprotein (HDL)-associated apolipoprotein), ATP-binding cassette transporter A1 (ABCA1), and scavenger receptor, class B, type I (SR-BI), proteins that promote cellular cholesterol mobilization. Liver X receptors (LXRs) and peroxisome-proliferator activated receptors (PPARs) are regulators of cholesterol transport, and activation of LXRs and PPARs has potential therapeutic implications for lipid-related neurodegenerative diseases. To clarify the functional impact of LXR/PPAR activation, sterol transport along the: (i) ABCA1/apoA-I and (ii) SR-BI/HDL pathway was investigated in primary, polarized brain capillary endothelial cells, an in vitro model of the blood-brain barrier. Activation of LXR (24(S)OH-cholesterol, TO901317), PPARalpha (bezafibrate, fenofibrate), and PPARgamma (troglitazone, pioglitazone) modulated expression of apoA-I, ABCA1, and SR-BI on mRNA and/or protein levels without compromising transendothelial electrical resistance or tight junction protein expression. LXR-agonists and troglitazone enhanced basolateral-to-apical cholesterol mobilization in the absence of exogenous sterol acceptors. Along with the induction of cell surface-located ABCA1, several agonists enhanced cholesterol mobilization in the presence of exogenous apoA-I, while efflux of 24(S)OH-cholesterol (the major brain cholesterol metabolite) in the presence of exogenous HDL remained unaffected. Summarizing, in cerebrovascular endothelial cells apoA-I, ABCA1, and SR-BI represent drug targets for LXR and PPAR-agonists to interfere with cholesterol homeostasis at the periphery of the central nervous system.     

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.     

Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells

ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.     

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.     

Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation

ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.     

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.     

ABCA1 promotes the de novo biogenesis of apolipoprotein CIII-containing HDL particles in vivo and modulates the severity of apolipoprotein CIII-induced hypertriglyceridemia

In this study, the ability of the lipid transporter ABCA1 and apolipoprotein CIII (apoCIII) to promote the de novo biogenesis of apoCIII-containing HDL in vivo and the role of this HDL in apoCIII-induced hypertriglyceridemia were investigated, using adenovirus-mediated gene transfer in apoE (-/-) x apoA-I (-/-) mice or ABCA1 (-/-) mice. Injection of apoE (-/-) x apoA-I (-/-) mice with 8 x 10 (8) pfu of an adenovirus expressing the wild-type human apoCIII (AdGFP-CIII g) generated HDL-like particles and triggered only a modest increase in plasma cholesterol and triglyceride levels of these mice, 3-5 days postinfection. Plasma human apoCIII was distributed among HDL, VLDL/IDL, and LDL in these mice. In contrast, ABCA1 (-/-) mice treated similarly failed to form HDL particles and developed severe hypertriglyceridemia which could be alleviated by coinfection with an adenovirus expressing human LpL, while their plasma cholesterol levels remained unchanged 3-5 days postinfection with AdGFP-CIII g. Human apoCIII in these mice accumulated exclusively on VLDL. Control experiments confirmed that the differences between apoE (-/-) x apoA-I (-/-) and ABCA1 (-/-) mice expressing human apoCIII were not due to differences in apoCIII expression. Overall, these data show that ABCA1 and human apoCIII promote the formation of apoCIII-containing HDL-like particles that are distinct from classical apoE- or apoA-I-containing HDL. Formation of apoCIII-containing HDL prevents excess accumulation of plasma apoCIII on VLDL and allows for the efficient lipolysis of VLDL triglycerides by LpL. Furthermore, the data establish that ABCA1 and apoCIII-containing HDL play key roles in the prevention of apoCIII-induced hypertriglyceridemia in mice.     

Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.     

Lysine residues of ABCA1 are required for the interaction with apoA-I

ATP-binding cassette protein A1 (ABCA1) plays a pivotal role in cholesterol homeostasis by generating high-density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), a lipid acceptor for ABCA1, reportedly interacts with ABCA1. However, it has also been proposed that apoA-I interacts with ABCA1-generated special domains on the plasma membrane, but apart from ABCA1, and solubilizes membrane lipids. To determine the importance of the apoA-I-ABCA1 interaction in HDL formation, the electrostatic interaction between apoA-I and ABCA1, which mediates the interaction between apoB100 in low-density lipoprotein particles (LDL) and LDL receptor, was analyzed. The apoA-I binding to ABCA1 and the cross-linking between them were inhibited by the highly charged molecules heparin and poly-L-lysine. Treating cells with membrane impermeable reagents that specifically react with primary amino groups abolished the interaction between apoA-I and ABCA1. However, these reagents did not affect the characteristic tight ATP binding to ABCA1. These results suggest that lysine residues in the extracellular domains of ABCA1 contribute to the interaction with apoA-I. The electrostatic interaction between ABCA1 and apoA-I is predicted to be the first step in HDL formation. This article is part of a Special Issue entitled Advances in high density lipoprotein formation and metabolism: a tribute to John F. Oram (1945-2010).     

Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that (125)I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Delta(187-243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of (125)I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of (125)I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 degrees C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.